Purified brush borders will be prepared from human intestinal mucosa obtained at surgery and post-mortem. The surface enzymes will be solublized by papain digestion and the solubilized peptide hydrolases will be separated by means of ion-exchange chromatography on DEAE- cellulose, preparative acrylamide-gel electrophoresis, and gel filtration chromatography. The substrate specificities of individual peptide hydrolases will be determined by means of a semi-quantitative high-voltage paper electrophoresis assay and subsequent quantitative assays. Other properties of the isolated enzymes including PH optima, stability, metal ion activation, and Michaelis constants will be determined. Cytoplasmic preparations obtained from the mucosa of fresh surgical intestinal specimens will be subjected to similar separation techniques. Isolated peptide hydrolases will be characterized in the same manner indicated for the brush border peptide hydrolases. Information obtained in the above studies will be used to determine the level of activity of specific brush border and cytoplasmic enzymes in per oral intestinal biopsies from patients with various diseases of undetermined etiology. The methods for measuring brush border and cytoplasmic peptide hydrolases in biopsy specimens will be the same as previously reported by this laboratory. In addition, further studies of the substrate specificity of certain brush border peptide hydrolases isolated from the rat intestinal mucosa will be carried out in an effort to obtain information which will make it possible to predict which peptides, among the hundreds not yet tested, are likely to be hydrolyzed by the 4 rat brush border peptide hydrolases previously isolated. Finally, studies of the rate of absorption of the amino and carboxy terminal amino acids of double- labeled peptides will be continued in an effort to further define the function of both the brush border and cytoplasmic peptide hydrolases.